The objective is to develop the use of fluorescence energy transfer to recover distance distributions between donors (D) and acceptors (A) which we are bound to biological macromolecules. During the first phase of this project we developed methods to recover distributions between donors and covalently-bound acceptors. These techniques were used for studies of D-A pairs linked by flexible alkyl chains, peptides of known sequence and rigidity, and to proteins in various conformational states. In the present proposal we intend to extend the methodology to the more complex case of multiple acceptors, which is of interest for lipid distributions in membranes and ion distributions around polyelectrolytes. Additionally, we will develop the theory and software to determine diffusive behavior of D-A systems. The information will be measured by both time-correlated single photon counting and by frequency-swept (1- 6000 MHz) phase-modulation fluorometry. Our specific goals are: 1. Determine random and non-random distributions of D-A pairs in two and three dimensions, such as in membranes and around membrane-bound proteins. 2. Determine end-to-end diffusion coefficients of D-A pairs linked by flexible alkyl chains, peptides of varying rigidity, helix- forming and Zn-finger peptides. 3. Develop measurements, theory, and software to recover non-random D-A distributions in one, two, and three dimensions. 4. Obtain enhanced resolution of D-A distance distributions by the development and use of global software for simultaneous analysis of both time and frequency-domain data, donor and acceptor decays, steady state data, and for global analysis with multiple Ro values. 5. Continue the present methods (single D-A pairs) to studies of conformation distributions of tRNA, calmodulin, and troponin. 6. Compare the experimentally recovered distance distributions o alkyl and peptide D-A pairs with those predicted by the rotational isomeric model or Monte Carlo calculations. 7. Compare the experimental and theoretical dependence of the energy transfer rate on kappa 2 using rigid indole-carbonyl D-A pairs.